I am interested in neuronal development and differentiation, with a particular interest in understanding the cellular and molecular mechanisms regulating neuronal ion channel expression. Currently, the research in my laboratory is focused on identifying the mechanisms regulating the expression of voltage-dependent sodium (Na) channels, key proteins in the production and propagation of action potentials. To accomplish this, we use a variety of molecular biological, biochemical, and electrophysiological approaches to analyze mammalian neurons, neuronal cell lines, and simple invertebrate nervous systems.

We have shown that an important aspect of Na channel regulation occurs at the level of gene expression. Large families of Na channel genes exist not only in mammals, but, as we have recently discovered, in simple invertebrate nervous systems as well. The individual members of these families exhibit distinct temporal and tissue-specifc patterns of expression, and appear to be selectively regulated during normal development as well as duirng regeneration and response to injury. Using nuclear run ons, Northern Blots, RNAse protection assays, and PCR as assays of gene expression, we are investigating the cellular and molecular mechanisms regulating the expression of the Na channel isoforms. In particular, we have begun to identify biochemical signaling pathways and mechanisms by which neurotrophic factors such as NGF and BDNF regulate Na channel gene expression in primary neurons and neuronal cell lines. The potential therapeutic value of neurotrophic factors for treatment of neurological disorders makes an understanding of their mechanisms of action of additional interest, and our expertise in this area has led to a number of collaborations with direct clinical implications. This includes functional analysis of novel mutations in RET receptor tyrosine kinases discovered by researchers at Dartmouth in patients with multple endocrine neoplasia, as well as analyses of the effects of neurotrophic factors on neurons from the mouse model of Niemann Pick Type C disease, which is characterized by deficits in cholestrol metabolism and, as we have recently discovered, a lack of growth factor responsiveness.

We are also analyzing the mechanisms governing Na channel expression at the level of subunit interactions, and have revealed a new level of complexity in the expression of these important membrane channels. Through analysis of epitope-tagged Na channel subunits in neuronal cell lines and primary neurons using immunofluorescence, Western blotting, and co-immunoprecipitation, we have revealed selectivity in the association of Na channel subunits, providing evidence for an additional level of complexity in the regulation of Na channel function and electrical excitability in an individual cell. In the process of generating and characterizing these tagged Na channel subunits, we also provided new information about the molecular moities involved in the interaction between subunits. We are continuing to exploit the use of these tagged subunits, as they should be useful for identifying additional proteins that interact with Na channels, analyzing posttranslational modifications such as phosphorylation that occur as part of Na channel regulation, and for providing new information about the subcellular localization of Na channels in excitable cells.

Finally, an important aspect of our efforts is to understand the functional consequences of the changes in Na channel expression we observe at the molecular biological and biochemical level, and we are investigating the role of specific Na channel subtypes in shaping the electrical membrane properties and signalling characteristics of excitable cells. Ths includes using single-cell PCR to analyze the expression of Na channel subtypes in simple invertebrate nervous systems composed of neurons with distinct patterns of electrical activity and well-defined physiological function. It also involves the use of patch clamp recording to analyze Na channel function in neuronal cell lines, primary neurons in culture, and in brain slices, whether under normal conditions or under experimental situations where we have manipulated the level of expression, cellular localization, or subunit composition of the Na channels in the membrane. In combination with the other molecular and biochemical approaches, this will ultimately help us understand the basis for neuronal excitability and electrical signaling in the nervous system.